Opioid receptors and methods of use

ABSTRACT

Genes encoding opioid receptors (including opioid-like receptor (ORL) proteins) can be retrieved from vertebrate libraries using the murine probe disclosed herein under low-stringency conditions. The DNA sequence shown in  FIG. 5  or its complement can be used to obtain the human delta, kappa and mu genes as well as the murine mu gene and human ORL-1. The probe provided encodes the murine delta opioid receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/823,114, filed 29Mar. 2001 and now abandoned, which is a continuation of U.S. Ser. No.09/148,351 filed 4 Sep. 1998 and now abandoned, which is a divisional ofU.S. Ser. No. 08/405,271 filed 14 Mar. 1995 and now U.S. Pat. No.6,432,652, which is a continuation-in-part of U.S. Ser. No. 08/403,260filed 13 May 1995 and now abandoned, which is a continuation-in-part ofU.S. Ser. No. 08/387,707 filed 13 Feb. 1995 and now issued as U.S. Pat.No. 6,265,563, which is the National Phase of PCT US 93/07665 filed 13Aug. 1993, which is a continuation-in-part of U.S. Ser. No. 07/929,200filed 13 Aug. 1992 and now abandoned. The contents of these applicationsare incorporated herein by reference.

This invention was made with Government support under Grant No. DA05010awarded by the Alcohol, Drug Abuse and Mental Health Administration. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

The invention relates to substances involved in vertebrate nervoussystems, and in particular to the opioid receptors and receptor-likeproteins (also referred to as opioid receptors herein) and activitiesmediated thereby. Accordingly, the invention concerns recombinantmaterials useful for the production of opioid receptors, the receptor asa diagnostic tool, therapeutic and diagnostic compositions relevant tothe receptor, and methods of using the receptor to screen for drugs thatmodulate the activity of the receptor.

BACKGROUND ART

The term “opioid” generically refers to all drugs, natural andsynthetic, that have morphine-like actions. Formerly, the term “opiate”was used to designate drugs derived from opium, e.g., morphine, codeine,and many semi-synthetic congeners of morphine. After the isolation ofpeptide compounds with morphine-like actions, the term opioid wasintroduced to refer generically to all drugs with morphine-like actions.Included among opioids are various peptides that exhibit morphine-likeactivity, such as endorphins, enkephalins and dynorphins. However, somesources have continued to use the term “opiate” in a generic sense, andin such contexts, opiate and opioid are interchangeable. Additionally,the term opioid has been used to refer to antagonists of morphine-likedrugs as well as to characterize receptors or binding sites that combinewith such agents.

Opioids are generally employed as analgesics, but they may have manyother pharmacological effects as well. Morphine and related opioidsproduce their major effects on the central nervous and digestivesystems. The effects are diverse, including analgesia, drowsiness, moodchanges, respiratory depression, dizziness, mental clouding, dysphoria,pruritus, increased pressure in the biliary tract, decreasedgastrointestinal motility, nausea, vomiting, and alterations of theendocrine and autonomic nervous systems.

A significant feature of the analgesia produced by opioids is that itoccurs without loss of consciousness. When therapeutic doses of morphineare given to patients with pain, they report that the pain is lessintense, less discomforting, or entirely gone. In addition toexperiencing relief of distress, some patients experience euphoria.However, when morphine in a selected pain-relieving dose is given to apain-free individual, the experience is not always pleasant; nausea iscommon, and vomiting may also occur. Drowsiness, inability toconcentrate, difficulty in mentation, apathy, lessened physicalactivity, reduced visual acuity, and lethargy may ensue.

The development of tolerance and physical dependence with repeated useis a characteristic feature of all opioid drugs, and the possibility ofdeveloping psychological dependence on the effect of these drugs is amajor limitation for their clinical use. There is evidence thatphosphorylation may be associated with tolerance in selected cellpopulations (Louie, A. et al. Biochem Biophys Res Comm (1988)152:1369-75).

Acute opioid poisoning may result from clinical overdosage, accidentaloverdosage, or attempted suicide. In a clinical setting, the triad ofcoma, pinpoint pupils, and depressed respiration suggest opioidpoisoning. Mixed poisonings including agents such as barbiturates oralcohol may also contribute to the clinical picture of acute opioidpoisoning. In any scenario of opioid poisoning, treatment must beadministered promptly.

The opioids interact with what appear to be several closely relatedreceptors. Various inferences have been drawn from data that haveattempted to correlate pharmacologic effects with the interactions ofopioids with a particular constellation of opioid receptors (Goodman andGilman's, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th ed, pp. 493-95(MacMillan 1985)). For example, analgesia has been associated with muand kappa receptors. Delta receptors are believed to be involved inalterations of affective behavior, based primarily on the localizationof these receptors in limbic regions of the brain. Additionally,activation, e.g., ligand binding with stimulation of furtherreceptor-mediated responses, of delta opioid receptors is believed toinhibit the release of other neurotransmitters. The pathways containingrelatively high populations of delta opioid receptor are similar to thepathways implicated to be involved in Huntington's disease. Accordingly,it is postulated that Huntington's disease may correlate with someeffect on delta opioid receptors.

Two distinct classes of opioid molecules can bind opioid receptors: theopioid peptides (e.g., the enkephalins, dynorphins, and endorphins) andthe alkaloid opiates (e.g., morphine, etorphine, diprenorphine andnaloxone). Subsequent to the initial demonstration of opiate bindingsites (Pert, C. B. and Snyder, S. H., Science (1973) 179:1011-1014), thedifferential pharmacological and physiological effects of both opioidpeptide analogues and alkaloid opiates served to delineate multipleopioid receptors. Accordingly, three anatomically and pharmacologicallydistinct opioid receptor types have been described: delta, kappa and mu.Furthermore, each type is believed to have sub-types (Wollemann, M., JNeurochem (1990) 54:1095-1101; Lord, J. A., et al., Nature (1977)267:495-499).

All three of these opioid receptor types appear share the samefunctional mechanisms at a cellular level. For example, the opioidreceptors cause inhibition of adenylate cyclase, and inhibition ofneurotransmitter release via both potassium channel activation andinhibition of Ca²⁺ channels (Evans, C. J., In: Biological Basis ofSubstance Abuse, S. G. Korenman & J. D. Barchas, eds., Oxford UniversityPress (in press); North, A. R., et al., Proc Natl Acad Sci USA (1990)87:7025-29; Gross, R. A., et al., Proc Natl Acad Sci USA (1990)87:7025-29; Sharma, S. K., et al., Proc Natl Acad Sci USA (1975)72:3092-96). Although the functional mechanisms are the same, thebehavioral manifestations of receptor-selective drugs differ greatly(Gilbert, P. E. & Martin, W. R., J Pharmacol Exp Ther (1976) 198:66-82).Such differences may be attributable in part to the anatomical locationof the different receptors.

Delta receptors have a more discrete distribution within the mammalianCNS than either mu or kappa receptors, with high concentrations in theamygdaloid complex, striatum, substantia nigra, olfactory bulb,olfactory tubercles, hippocampal formation, and the cerebral cortex(Mansour, A., et al., Trends in Neurosci (1988) 11:308-14). The ratcerebellum is remarkably devoid of opioid receptors including deltaopioid receptors.

Several opioid molecules are known to selectively or preferentially binddelta receptors. Of the vertebrate endogenous opioids, the enkephalins,particularly met-enkephalin (SEQ ID NO: 1) and leu-enkephalin (SEQ IDNO: 2), appear to possess the highest affinity for delta receptors,although the enkephalins also have high affinity for mu receptors.Additionally, the deltorphans, peptides isolated from frog skin,comprise a family of opioid peptides that have high affinity andselectivity for delta receptors (Erspamer, V., et al., Proc Natl AcadSci USA (1989) 86:5188-92).

A number of synthetic enkephalin analogues are also deltareceptor-selective including (D-Ser²) leucine enkephalin Thr (DSLET)(SEQ ID NO: 3) (Garcel, G. et al. FEBS Lett (1980) 118:245-247) and(D-Pen², D-Pen⁵) enkephalin (DPDPE) (SEQ ID NO: 4) (Akiyama, K. et al.,Proc Natl Acad Sci USA (1985) 82:2543-2547).

Recently a number of other selective delta receptor ligands have beensynthesized, and their bioactivities and binding characteristics suggestthe existence of more than one delta receptor subtype (Takemori, A. E.,et al., Ann Rev Pharm Toxicol, (1992) 32:239-69; Negri, L., et al., EurJ Pharmacol (1991) 196:355-335; Sofuoglu, M., et al., Pharmacologist(1990) 32:151).

Although the synthetic pentapeptide 2dAla, 5dLeu enkephalin (DADLE) wasconsidered to be delta-selective, it also binds equally well to mureceptors. The synthetic peptide D-Ala²-N-Me-Phe⁴-Gly-ol⁵-enkephalin(DAGO) (SEQ ID NO: 6) has been found to be a selective ligand formu-receptors.

The existence of multiple delta opioid receptors has been implied notonly from the pharmacological studies addressed above, but also frommolecular weight estimates obtained by use of irreversible affinityligands. Molecular weights for the delta opioid receptor that range from30 kDa to 60 kda (Evans, C. J., supra; Evans, C. J. et al., Science(1992) 258:1952-1955, which document corresponds to the disclosure ofthe priority document of the present application; Bochet, P. et al., MolPharmacol (1988) 34:436-43). The various receptor sizes may representalternative splice products, although this has not been established.

Many studies of the delta opioid receptor have been performed with theneuroblastoma/glioma cell line NG108-15, which was generated by fusionof the rat glial cell line (C6BU-1) and the mouse neuroblastoma cellline (N18-TG2) (Klee, W. A. and Nirenberg, M. A., Proc Natl Acad Sci USA(1974) 71:3474-3477). The rat glial cell line expresses essentially nodelta opioid receptors, whereas the mouse neuroblastoma cell lineexpresses low amounts of the receptor. Thus, it has been suggested thatthe delta receptor in the NG108-15 cells is of mouse chromosomal origin(Law, Mol Pharm (1982) 21:438-91). Each NG108-15 cell is estimated toexpress approximately 300,000 delta-receptors. Only delta-type opioidreceptors are expressed, although it is not known whether theserepresent more than a single subtype. Thus, the NG108-15 cell line hasserved to provide considerable insight into the binding characterizationof opioid receptors, particularly delta opioid receptors. However, theNG108-15 cell line is a cancer-hybrid and may not be completelyrepresentative of the delta receptor in endogenous neurons due to theunique cellular environment in the hybrid cells.

An extensive literature has argued that the opioid receptors are coupledto G-proteins (see, e.g., Schofield, P. R., et al., EMBO J (1989)8:489-95), and are thus members of the family of G-protein coupledreceptors. G-proteins are guanine nucleotide binding proteins thatcouple the extracellular signals received by cell surface receptors tovarious intracellular second messenger systems. Identified members ofthe G-protein-coupled family share a number of structural features, themost highly conserved being seven apparent membrane-spanning regions,which are highly homologous among the members of this family (Strosberg,A. D., Eur J Biochem (1991) 196:1-10). Evidence that the opioidreceptors are members of this family includes the stimulation of GTPaseactivity by opioids, the observation that GTP analogues dramaticallyeffect opioid and opiate agonist binding, and the observation thatpertussis toxin (which by ADP ribosylation selectively inactivates boththe Gi and Go subfamilies of G-proteins) blocks opioid receptor couplingto adenylate cyclase and to K⁺ and Ca²⁺ channels (Evans, C. J., supra).

The members of the G-protein-coupled receptor family exhibit a range ofcharacteristics. Many of the G-protein-coupled receptors, e.g., thesomatostatin receptor and the angiotensin receptor, have a single exonthat encodes the entire protein coding region (Strosberg supra; Langord,K., et al., Biochem Biophys Res Comm (1992) 138:1025-1032). However,other receptors, such as substance P receptor and the dopamine D-2receptor, contain the protein coding region. The D-2 receptor isparticularly interesting in that alternate splicing of the gene givesrise to different transcribed products (i.e., receptors) (Evans, C. J.,supra; Strosberg, supra). Interestingly, somatostatin ligands arereported to bind to opioid receptors (Terenius, L., Eur J Pharmacol(1976) 38:211; Mulder, A. H., et al., Eur J Pharmacol (1991) 205:1-6)and, furthermore, to have similar molecular mechanisms (Tsunoo, A., etal., Proc Natl Acad Sci USA (1986) 83:9832-9836).

In previous efforts to describe and purify opioid receptors, two cloneshave been described that were hypothesized either to encode a portion ofor entire opioid receptors. The first clone, which encodes the opiatebinding protein OBCAM (Schofield et al., supra), was obtained byutilizing a probe designed from an amino acid sequence of a proteinpurified on a morphine affinity column. OBCAM lacks any membranespanning domains but does have a C-terminal domain that ischaracteristic of attachment of the protein to the membrane by aphosphatidylinositol (PI) linkage. This feature, which is shared bymembers of the immunoglobulin superfamily, is not common to the familyof receptors coupled to G-proteins. Thus, it has been proposed thatOBCAM is part of a receptor complex along with other components that arecoupled to G-proteins (Schofield et al., supra). At present, however,there is no direct evidence for such a complex.

A second proposed opioid receptor clone was obtained in an effort toclone a receptor that binds kappa opioid receptor ligands (Xie, G. X.,Proc Natl Acad Sci USA (1992) 89:4124-4128). A DNA molecule encoding aG-coupled receptor from a placental cDNA library was isolated. Thisreceptor has an extremely high homology with the neurokinin B receptor(84% identical throughout the proposed protein sequence). When thisclone was expressed in COS cells, it displayed opioid peptidedisplaceable binding of ³H-bremazocine (an opiate ligand with highaffinity for kappa receptors). However, the low affinity of thisreceptor for ³H-bremazocine, and the lack of appropriate selectivitysince this receptor (binding both mu and delta ligands) made it doubtfulthat this cloned molecule is actually an opioid receptor.

Furthermore, characterization of opioid receptor proteins has provendifficult because of their instability once solubilized from themembrane; purified delta opioid receptors have not been isolated. Theprevious estimates of opioid receptor molecular weights ranging from 30kDa to 60 Kda further reflect the difficulty in isolating andcharacterizing these proteins.

Recently, DNA encoding the murine kappa and delta opioid receptors frommouse brain was reported by Yasuda, K. et al. Proc Natl Acad Sci USA(1993) 90:6736-6740. The sequence of the clones indicated the presenceof the expected seven transmembrane regions. In addition, Chen, Y. etal. in a soon-to-be-published manuscript in Molecular Pharmacology(1993) report the “molecular cloning and functional expression of a muopioid receptor from rat brain”. In fact, the rat mu receptor was clonedusing the present inventors' DOR-1 clone, which lends enabling supportto the present invention disclosed below. The mouse delta opioidreceptor was disclosed as having been cloned (Kieffer, B. J. et al.,Proc Natl Acad Sci USA (1992) 89:12048-12052 (December issue) after thefiling date of the priority document of the present application.However, the sequence reported therein differs from the sequencereported by the present inventors for the mouse delta receptor (Evans etal., 1992, supra; this disclosure).

In addition to the opioid receptors which respond to specified agonists,the delta, kappa and mu opioid receptors, additional forms of theseproteins, commonly called opioid receptor-like (ORL) proteins have beenobtained using the methods described herein. Using these methods, twohuman ORL protein-encoding cDNAs were obtained from a human brain stemcDNA library. One of these clones is equivalent to that isolated byO'Dowd, B. F. et al. Gene (1993) 136:355-360; the other, ORL-1, isidentical to that reported by Mollereau, C. et al. FEBS Lett (1994)341:33-38. A preliminary report of the present work appeared inRegulatory Peptides (1994) 54:143-144 and is incorporated herein byreference.

DISCLOSURE OF THE INVENTION

The present invention provides recombinant nucleic acid molecules whichencode the murine delta opioid receptor, as well as recombinant nucleicacid molecules which can be retrieved using low-stringency hybridizationto this disclosed DNA. Thus, the invention provides genes encoding thedelta, kappa and mu receptors, representing opioid receptors generally,including ORL proteins, of any species containing genes encoding suchreceptors or ORL proteins sufficiently homologous to hybridize underlow-stringency conditions described herein.

As used herein, “opioid receptors” includes not only the previouslyidentified delta, kappa and mu receptors, but also additionalreceptor-like proteins, represented by, for example, ORL-1 thathybridize under the low-stringency conditions described to the murineDOR clone set forth herein, and which have opioid receptorcharacteristics including seven putative transmembrane regions, andability to couple with guanine nucleotide-binding regulatory proteins (Gproteins) to inhibit adenylyl cyclase and/or calcium channels or tostimulate potassium channels. Thus, when the word “opioid receptors” isused hereinbelow, this term is intended to include this entire genus.

Thus, in one aspect, the invention is directed to recombinant nucleicacid molecules and methods for the production of an opioid receptorwherein the opioid receptor is encoded by a gene which hybridizes underlow-stringency to the nucleotide sequence of FIG. 5 or to itscomplement. By “low-stringency” is meant 50% formamide/6×SSC, overnightat 37° C. for the hybridization, followed by washes at 2×SSC 0.1% SDS atroom temperature or 50% formamide/6×SSC at 37° C. with washes of1×SSC/0.1% SDS at 37° C.

Also provided are expression systems comprising the nucleic acidmolecules described above. The receptor can be recombinantly producedusing these expression systems and host cells modified to contain them.

Especially useful are vertebrate cells which express the opioid receptorgene so that the opioid receptor protein is displayed at the surface ofthe cells. These cells offer means to screen native and syntheticcandidate agonists and antagonists for the opioid receptors.

In still other aspects, the invention is directed to methods to screencandidate agonists and/or antagonists acting at opioid receptors usingthe recombinant transformed cells of the invention. Such assays include(1) binding assays using competition with ligands known to bind opioidreceptors, (2) agonist assays which analyze activation of the secondarypathways associated with opioid receptor activation in the transformedcells, and (3) assays which evaluate the effect on binding of thecandidate to the receptor by the presence or absence of sodium ion andGTP. Antagonist assays include the combination of the ability of thecandidate to bind the receptor while failing to effect furtheractivation, and, more importantly, competition with a known agonist.

Still another aspect of the invention is provision of antibodycompositions which are immunoreactive with the opioid receptor proteins.Such antibodies are useful, for example, in purification of thereceptors after solubilization or after recombinant production thereof.

In still other aspects, the invention is directed to probes useful forthe identification of DNA which encodes related opioid receptors invarious species or different types and subtypes of opioid receptors.

Accordingly, an object of the present invention is to provide anisolated and purified form of a DNA sequence encoding an opioidreceptor, which is useful as a probe as well as in the production of thereceptor.

Another object is to provide a recombinantly produced DNA sequenceencoding an opioid receptor.

Another object is to produce an antisense sequences corresponding toknown sense sequences encoding the opioid receptors.

Another object of the invention is to provide a DNA construct comprisedof a control sequence operatively. linked to a DNA sequence whichencodes an opioid receptor and to provide recombinant host cellsmodified to contain the DNA construct.

Another object is to isolate, clone and characterize, from variousvertebrate species, DNA sequences encoding the various relatedreceptors, by hybridization of the DNA derived from such species with anative DNA sequence encoding the opioid receptor of the invention.

An advantage of the present invention is that opioid receptor-encodingDNA sequences can be expressed at the surface of host cells which canconveniently be used to screen drugs for their ability to interact withand/or bind to the receptors.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of binding of ³H-diprenorphine (saturationcurves) between NG108-15 cells and COS cells three days followingtransfection (by electroporation) of each with DOR-1 in the CDM-8vector. Specific opioid binding was undetectable in nontransfected COScells or COS cells transfected with plasmid alone.

FIG. 2 depicts displacement curves of 5 nM ³H-diprenorphine from COScell membranes of cells transfected with DOR-1. ³H-diprenorphine wasdisplaced by diprenorphine, etorphine, morphine and levorphanol, but notby dextrorphan (the non-opiate active optical isomer of levorphanol).

FIG. 3 depicts displacement curves of 5 nm ³H-diprenorphine from COScell membranes of cells transfected with DOR-1. ³H-diprenorphine wasdisplaced by DPDPE and DSLET, which are delta-selective agonists, byDADLE, a high affinity ligand for mu and delta receptors, and bydynorphin 1-17, a kappa-preferring ligand. ³H-diprenorphine was notdisplaced by DAGO, a mu-selective ligand.

FIG. 4 depicts the results of a Northern analysis of mRNA from NG108-15cells and cells from various rat brain regions.

FIGS. 5 a and 5 b shows the nucleotide sequence and the deduced aminoacid sequence of the DOR-1 clone (SEQ ID NO: 7).

FIG. 6 depicts the deduced amino acid sequence of DOR-1 (SEQ ID NO: 8),compared with the rat somatostatin receptor. Consensus glycosylationsites predicted to fall in extracellular domains are indicated by anasterisk. Potential protein kinase C sites are listed in Example 5. Theseven predicted membrane spanning regions (underlined) are predictedbased on the hydrophobicity profile and published predictions (MacVectorsoftware program (IBI); T. Hopp, and K. Woods, Proc Natl Acad Sci USA(1981) 78:3842-3828). For sequencing, the cDNA insert was subcloned intopBluescript and both strands were sequenced from single-stranded DNAusing Sequenase and Taq cycle sequencing. For ambiguities due tocompressions 7-deaza-dGTP replaced dGTP in the sequencing reactions andthe products were resolved on formamide gels.

FIG. 7 depicts a Southern blot of radiolabeled DOR-1 cDNA probehybridized at high stringency to NG108-15, mouse, rat and human DNA cutwith BamHI.

FIG. 8 a shows a partial nucleotide sequence of the human delta opioidreceptor genomic clone H3 (also designated human DORa or hDORa). (SEQ IDNOs: 10 and 11)

FIG. 8 b-1-8 b-4 shows a partial nucleotide sequence of the human kappaopioid receptor genomic clone H14 (also designated human KORa or hKORa).(SEQ ID NO: 12)

FIG. 8 c-1-8 c-2 shows a partial nucleotide sequence of the human muopioid receptor genomic clone H20 (also designated human MORa or hMORa).(SEQ ID NO: 13)

FIG. 8 d shows the nucleotide sequence of the CACACA repeat near the H₂₀DNA. (SEQ ID NO: 14)

FIG. 9 a-9 c shows the nucleotide sequence of the murine mu-receptorclone DOR-2 also named mMOR-1 or mMOR-1α. (SEQ ID NO: 15)

FIG. 10 a-10 b shows the homology of various receptor amino acidsequences. (SEQ ID NO: 8, 16 and 17)

FIG. 11A-11C shows the complete DNA sequence of the cDNA retrieved fromhuman brain stem cDNA library and comprising a nucleotide sequenceencoding the opioid receptor ORL-1. (SEQ ID NO: 18) This cDNA encodes a370-amino acid opioid receptor protein. (SEQ ID NO: 19)

FIG. 12A-12B shows a comparison of ORL-1 and ORL-2 amino acid sequenceswith various human and murine delta, kappa, and mu receptors. (SEQ IDNOs: 20-24) ORL-1 is a protein of 370 amino acids and is compared withhuman mu opioid receptor (hMOR), human delta opioid receptor (hDOR) andmurine kappa opioid receptor (mKOR).

MODES OF CARRYING OUT THE INVENTION

The invention provides DNA encoding mammalian opioid receptor proteinand additional recombinant nucleic acids, expression vectors and methodsuseful for the production of these proteins. In addition, eucaryoticcells, such as COS cells, transformed with the recombinant molecules ofthe invention so as to express opioid receptor proteins at their surfaceare useful in screening assays to identify candidate opioid agonists andantagonists. In addition, antibodies may be raised to the recombinantlyproduced opioid receptor proteins. These antibodies are useful inimmunoassays for said protein and in affinity purification thereof.

Recombinant Opioid Receptor

Illustrated hereinbelow is the obtention of a cDNA encoding a murinedelta opioid receptor. The complete DNA sequence of the cDNA, and theamino acid sequence encoded thereby, are set forth herein in FIG. 5. Theavailability of this cDNA permits the retrieval of the correspondingopioid receptor-encoding DNA from other vertebrate species. Accordingly,the present invention places within the possession of the art,recombinant molecules and methods for the production of cells expressingopioid receptors of various types and of various vertebrate species.Thus, the cDNA of FIG. 5, or a portion thereof, may be used as a probeto identify that portion of vertebrate genomic DNA or cDNA which encodesan opioid receptor protein. Illustrative methods used to prepare agenomic library and identify the opioid receptor-encoding genes aredescribed for convenience hereinbelow. Also exemplified as illustratingthe method of the invention is the retrieval of human ORL-1 from a brainstem cDNA library.

The DOR-1 clone described in FIG. 5 is a cDNA clone corresponding to themurine delta opioid receptor. The present inventors found, and describeherein, that screening of a human genomic library under conditions oflow stringency results in the recovery of DNA encoding all three typesof human opioid receptors. Similarly, a murine genomic clone wasobtained. In addition, a cDNA clone was obtained from a mouse brainlibrary encoding the murine mu opioid receptor. Thus, either cDNAlibraries from appropriate sources, such as brain, or genomic libraries,are fruitful sources or substrates for obtaining the DNA of the presentinvention and the corresponding recombinant materials. The invention isthus directed to DNA encoding an opioid receptor of a vertebrate,wherein the opioid receptor is encoded by a nucleotide sequence whichhybridizes under conditions of low stringency to the nucleotide sequenceshown in FIG. 5 or to its complement.

In the alternative, the DNA of FIG. 5 or a portion thereof may be usedto identify specific tissues or cells which express opioid receptorprotein by analyzing the mRNA, for example, using Northern blottechniques. Those tissues which are identified as containing mRNAencoding opioid receptor protein using the probes of the invention arethen suitable sources for preparation of cDNA libraries which mayfurther be probed using the cDNA described hereinbelow.

The DNA encoding the various vertebrate opioid receptor proteins,obtained in general as set forth above, according to the standardtechniques described hereinbelow, can be used to produce cells whichexpress the opioid receptor at their surface; such cells are typicallyeucaryotic cells, in particular, mammalian cells such as COS cells orCHO cells. Suitable expression systems in eucaryotic cells for suchproduction are described hereinbelow. The opioid receptor proteins mayalso be produced in procaryotes or in alternative eucaryotic expressionsystems for production of the protein per se. The DNA encoding theprotein may be ligated into expression vectors preceded by signalsequences to effect its secretion, or may be produced intracellularly,as well as at the cell surface, depending on the choice of expressionsystem and host. If desired, the opioid receptor protein thusrecombinantly produced may be purified using suitable means of proteinpurification, and, in particular, by affinity purification usingantibodies or fragments thereof immunospecific for the opioid receptorprotein.

The reader is reminded that the term “opioid receptor” as used hereinincludes not only the conventional delta, kappa and mu opioid receptors,but also opioid receptor-like proteins which interact with G proteins ina similar manner. These receptor-like proteins are useful in analogousways, and offer additional screening tools for candidate compounds thataffect the central nervous system. They are thus useful for the samepurposes as the “conventional” receptors.

Screening for Opioid Agonists and Antagonists Using Recombinant Cells

The ability of a candidate compound to act as an opioid agonist orantagonist may be assessed using the recombinant cells of the inventionin a variety of ways. To exhibit either agonist or antagonist activity,the candidate compound must bind to the opioid receptor. Thus, to assessthe ability of the candidate to bind, either a direct or indirectbinding assay may be used. For a direct binding assay, the candidatebinding compound is itself detectably labeled, such as with aradioisotope or fluorescent label, and binding to the recombinant cellsof the invention is assessed by comparing the acquisition of label bythe recombinant cells to the acquisition of label by correspondinguntransformed (control) cells.

More convenient, however, is the use of a competitive assay wherein thecandidate compound competes for binding to the recombinant cells of theinvention with a detectably labeled form of an opioid ligand known tobind to the receptor. Such ligands are themselves labeled usingradioisotopes or fluorescent moieties, for example. A particularlysuitable opioid known to bind to this receptor is diprenorphine. Atypical protocol for such an assay is as follows:

In general, about 10⁶ recombinant cells are incubated in suspension in1.0 ml of Kreb's Ringer Hepes Buffer (KRHB) at pH 7.4, 37° C. for 20 minwith ³H-diprenorphine. Nonspecific binding is determined by the additionof 400 nM diprenorphine in the binding mixtures. Various concentrationsof candidate compounds are added to the reaction mixtures. Theincubations are terminated by collecting the cells on Whatman GF-Bfilters, with removal of excess radioactivity by washing the filtersthree times with 5 ml of KRHB at 0° C. After incubating at 20° C.overnight in 5 ml of scintillation fluid, such as Liquiscint (NationalDiagnostics, Somerville, N.J.), the radioactivity on the filters isdetermined by liquid scintillation counting.

The K_(d) (dissociation constant) values for the candidate opiateligands can be determined from the IC₅₀ value (“inhibitoryconcentration₅₀” means the concentration of candidate ligand thatresults in a 50% decrease in binding of labeled diprenorphine).

The effects of sodium and GTP on the binding of ligands to therecombinantly expressed receptors can be used to distinguish agonistfrom antagonist activities. If the binding of a candidate compound issensitive to Na⁺ and GTP, it is more likely to be an agonist than anantagonist, since the functional coupling of opioid receptors to secondmessenger molecules such as adenylate cyclase requires the presence ofboth sodium and GTP (Blume et al., Proc Natl Acad Sci USA (1979)73:26-35). Furthermore, sodium, GTP, and GTP analogues have been shownto effect the binding of opioids and opioid agonists to opioid receptors(Blume, Life Sci (1978) 22:1843-52). Since opioid antagonists do notexhibit binding that is sensitive to guanine nucleotides and sodium,this effect is used as a method for distinguishing agonists fromantagonists using binding assays.

In addition, agonist activity can directly be assessed by the functionalresult within the cell. For example, it is known that the binding ofopioid agonists inhibits cAMP formation, inhibits potassium channelactivation, inhibits calcium channel activation, and stimulates GTPase.Assessment of these activities in response to a candidate compound isdiagnostic of agonist activity. In addition, the ability of a compoundto interfere with the activating activity of a known agonist such asetorphine effectively classifies it as an antagonist.

In one typical assay, the measurement of cAMP levels in cells expressingopioid receptors is carried out by determining the amount of ³H-cAMPformed from intracellular ATP pools prelabeled with ³H-adenine (Law etal., supra). Thus, cAMP formation assays are carried out with 0.5×10⁶cells/0.5 ml of KRHB at pH 7.4, incubated at 37° C. for 20 minutes.After addition of the internal standard ³²P-cAMP, the radioactive cAMPis separated from other ³H-labeled nucleotides by known double-columnchromatographic methods. The opiate agonists' ability to inhibit cAMPaccumulation is then determined as described by Law et al. (supra).

The potency of a candidate opiate antagonist can be determined bymeasuring the ability of etorphine to inhibit cyclic AMP accumulation inthe presence and in the absence of known amounts of the candidateantagonist. The inhibition constant (K_(i)) of an antagonist can then becalculated from the equation or competitive inhibitors.

An interesting feature of screening assays using the prior art NG108-15cells is that the agonist adenylate cyclase inhibition functionapparently does not require binding of all receptors on these cells.Thus, the K_(d) and K_(i) values for the opioid ligands differed whenusing these cells.

The foregoing assays, as described above, performed on the recombinantlytransformed cells of the present invention, provide a more direct andmore convenient screen for candidate compounds having agonist andantagonist opioid receptor activity than that previously available inthe art. Furthermore, such assays are more sensitive since cells can, inaccordance with the present invention, be engineered to express highlevels of the opioid receptor. Additionally, cells engineered inaccordance with the present invention will circumvent the concern thatNG108-15 cells, due to their tumor cell background, have a cellularenvironment that artifactually affects opioid receptor expression.

The mu opioid encoding DNA described herein also offer a means to followinheritance patterns. DNA sequence polymorphisms frequently occur in thenoncoding regions that surround genes. Polymorphisms are especiallyfrequent in repeat sequences such as CACACA which often show distinctpolymorphisms in the number of repeats that are present in differentindividuals. These polymorphisms offer a marker by which to follow theinheritance of the gene among family members. The inheritance of a gene(such as MORa) or its human counterpart can be followed by polymerasechain reaction (PCR) amplification of the region surrounding the CACACApolymorphism and analyzing the resulting products. This would be auseful diagnostic marker for the mu opioid receptor gene.

Methods to Prepare Opioid Receptor Protein or Portions Thereof

The present invention provides the amino acid sequence of a murineopioid receptor; similarly, the availability of the cDNA of theinvention places within possession of the art corresponding vertebrateopioid receptors whose amino acid sequence may also be determined bystandard methods. As the amino acid sequences of such opioid receptorsare known, or determinable, in addition to purification of such receptorprotein from native sources, recombinant production or synthetic peptidemethodology may also be employed for producing the receptor protein orpeptide.

The opioid receptor or portions thereof can thus also be prepared usingstandard solid phase (or solution phase) peptide synthesis methods, asis known in the art. In addition, the DNA encoding these peptides may besynthesized using commercially available oligonucleotide synthesisinstrumentation for production of the protein in the manner set forthabove. Production using solid phase peptide synthesis is, of course,required if amino acids not encoded by the gene are to be included.

The nomenclature used to describe the peptides and proteins of theinvention follows the conventional practice where the N-terminal aminogroup is assumed to be to the left and the carboxy group to the right ofeach amino acid residue in the peptide. In the formulas representingselected specific embodiments of the present invention, the amino- andcarboxy-terminal groups, although often not specifically shown, will beunderstood to be in the form they would assume at physiological pHvalues, unless otherwise specified. Thus, the N-terminal NH3⁺ andC-terminal COO⁻ at physiological pH are understood to be present thoughnot necessarily specified and shown, either in specific examples or ingeneric formulas. Free functional groups on the side chains of the aminoacid residues may also be modified by glycosylation, phosphorylation,cysteine binding, amidation, acylation or other substitution, which can,for example, alter the physiological, biochemical, or biologicalproperties of the compounds without affecting their activity within themeaning of the appended claims.

In the peptides shown, each gene-encoded residue, where appropriate, isrepresented by a single letter designation, corresponding to the trivialname of the amino acid, in accordance with the following conventionallist:

One-Letter Three-letter Amino Acid Symbol Symbol Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V ValNomenclature of Enkephalins

Enkephalins are either of two peptides having five residues with theN-terminal residue numbered 1:

tyr-gly-gly-phe-xxx (SEQ ID NO: 25)  1   2   3   4   5In “met enkephalin” the fifth residue is methionine:

-   -   tyr-gly-gly-phe-met (SEQ ID NO: 1)        In “leu enkephalin” the 5th residue is leucine:    -   tyr-gly-gly-phe-leu (SEQ ID NO: 2)        Enkephalin analogs can be made with (1) amino acid        substitutions, (2) D-amino acid substitutions, and/or (3)        additional amino acids. The site at which the substitution is        made is noted at the beginning of the compound name. For        example, “(D-ala², D-leu⁵) enkephalin” means that D-ala is        present at the second position and D-leu is present at the fifth        position:    -   tyr-[D-ala]-gly-phe-[D-leu] (SEQ ID NO: 5)        One letter abbreviations can also be used. Thus, “(D-ser²) leu        enkephalin” could be abbreviated as “DSLE.” Additional residues        are noted as well. Thus, the addition of a threonine residue (to        the sixth position) of (D-ser²) leu enkephalin would be        “(D-ser²) leu enkephalin thr” which could be abbreviated as        “DSLET”:    -   tyr-[D-ser]-gly-phe-leu-thr (SEQ ID NO: 3)        Antibodies

Antibodies immunoreactive with the opioid receptor protein or peptide ofthe present invention can be obtained by immunization of suitablemammalian subjects with peptides, containing as antigenic regions thoseportions of the receptor intended to be targeted by the antibodies.Certain protein sequences have been determined to have a high antigenicpotential. Such sequences are listed in antigenic indices, for example,Macvector software (I.B.I.) Thus, by determining the sequence of theopioid receptor protein and evaluating the sequence with an antigenicindex, probable antigenic sequences are located.

Antibodies are prepared by immunizing suitable mammalian hosts accordingto known immunization protocols using the peptide haptens alone, if theyare of sufficient length, or, if desired, or if required to enhanceimmunogenicity, conjugated to suitable carriers. Methods for preparingimmunogenic conjugates with carriers such as BSA, KLH, or other carrierproteins are well known in the art. In some circumstances, directconjugation using, for example, carbodiimide reagents may be effective;in other instances linking reagents such as those supplied by PierceChemical Co., Rockford, Ill., may be desirable to provide accessibilityto the hapten. The hapten peptides can be extended or interspersed withcysteine residues, for example, to facilitate linking to carrier.Administration of the immunogens is conducted generally by injectionover a suitable time period and with use of suitable adjuvants, as isgenerally understood in the art. During the immunization schedule,titers of antibodies are taken to determine adequacy of antibodyformation.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal antibody (mAb) preparations is preferred. Immortalized celllines which secrete the desired mAbs may be prepared using the standardmethod of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired mAbs are screened byimmunoassay in which the antigen is the peptide hapten or is the opioidreceptor itself displayed on a recombinant host cell. When theappropriate immortalized cell culture secreting the desired mAb isidentified, the cells can be cultured either in vitro or byintraperitoneal injection into animals wherein the mAbs are produced inthe ascites fluid.

The desired mAbs are then recovered from the culture supernatant or fromthe ascites fluid. In addition to intact antibodies, fragments of themAbs or of polyclonal antibodies which contain the antigen-bindingportion can be used as antagonists. Use of immunologically reactiveantigen binding fragments, such as the Fab, Fab′, of F(ab′)₂ fragments,is often preferable, especially in a therapeutic context, as thesefragments are generally less immunogenic than the whole immunoglobulinmolecule.

Standard Methods

The techniques for sequencing, cloning and expressing DNA sequencesencoding the amino acid sequences corresponding to a opioid receptor,e.g., polymerase chain reaction (PCR), synthesis of oligonucleotides,probing a cDNA library, transforming cells, constructing vectors,preparing antisense oligonucleotide sequences based on known sensenucleotide sequences, extracting messenger RNA, preparing cDNAlibraries, and the like are well-established in the art. Ordinarilyskilled artisans are familiar with the standard resource materials,specific conditions and procedures. The following paragraphs areprovided for convenience, it being understood that the invention islimited only by the appended claims.

RNA Preparation and Northern Blot

RNA preparation is as follows: The samples used for preparation of RNAare immediately frozen in liquid nitrogen and then stored until use at−80° C. The RNA is prepared by CsCl centrifugation (Ausubel et al.,supra) using a modified homogenization buffer (Chirgwin et al.,Biochemistry (1979) 18:5294-5299). Poly(A⁺) RNA is selected by oligo(dT)chromatography (Aviv and Leder, Proc Natl Acad Sci USA (1972)69:1408-1412). RNA samples are stored at −80° C.

Analysis of gene expression and tissue distribution can be accomplishedusing Northern blots with, e.g., radiolabeled probes. The mRNA issize-separated using gel electrophoresis and then typically istransferred to a nylon membrane or to nitrocellulose and hybridized withradiolabeled probe. Presence of the hybridized probe is detected usingautoradiography.

Cloning

The cDNA sequences encoding the opioid receptor protein are obtainedfrom a random-primed, size-selected cDNA library.

Alternatively, the cDNA sequences encoding opioid receptor protein areobtained from a cDNA library prepared from mRNA isolated from cellsexpressing the receptor protein in various organs such as the brain,according to procedures described in Sambrook, J. et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y., 1989.

The cDNA insert from the successful clone, excised with a restrictionenzyme such as EcoRI, is then used as a probe of the original cDNAlibrary or other libraries (low stringency) to obtain the additionalclones containing inserts encoding other regions of the protein thattogether or alone span the entire sequence of nucleotides coding for theprotein.

An additional procedure for obtaining cDNA sequences encoding the opioidreceptor protein is PCR. PCR is used to amplify sequences from a pooledcDNA library of reversed-transcribed RNA, using oligonucleotide primersbased on the transporter sequences already known.

Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employs ligation and restriction techniques which arewell understood in the art (Young et al., Nature (1988) 316:450-452).Double-stranded cDNA encoding opioid receptor protein is synthesized andprepared for insertion into a plasmid vector CDM8. Alternatively,vectors such as Bluescript² or Lambda ZAP² (Stratagene, San Diego,Calif.) or a vector from Clontech (Palo Alto, Calif.) can be used inaccordance with standard procedures (Sambrook, J. et al., supra)

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme, such as EcoRI, or more than one enzyme, underconditions which are generally understood in the art, and theparticulars of which are specified by the manufacturer of thesecommercially available restriction enzymes. See, e.g., New EnglandBiolabs, Product Catalog. In general, about 1 μg of DNA is cleaved byone unit of enzyme in about 20 μl of buffer solution; in the examplesherein, typically, an excess of restriction enzyme is used to ensurecomplete digestion of the DNA substrate. Incubation times of about oneto two hours at about 37° C. are workable, although variations can betolerated. After each incubation, protein is removed by extraction withphenol/chloroform, and can be followed by other extraction and thenucleic acid recovered from aqueous fractions by precipitation withethanol.

In vector construction employing “vector fragments”, the vector fragmentis commonly treated with bacterial alkaline phosphatase (BAP) or calfintestinal alkaline phosphatase (CIP) in order to remove the 5′phosphate and prevent religation of the vector. Digestions are conductedat pH 8 in approximately 150 mM Tris, in the presence of Na⁺ and Mg⁺⁺using about 1 unit of BAP or CIP per μg of vector at 60° C. or 37° C.,respectively, for about one hour. In order to recover the nucleic acidfragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors is which have been double digested by additional restrictionenzyme digestion of the unwanted fragments.

Ligations are performed in 15-50 μl volumes under the following standardconditions and temperatures: 20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM to 50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 0° C. (for “sticky end” ligation) or 1 mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for “blunt end”ligation). Intermolecular “sticky end” ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers) are performed at 1 μM total endsconcentration. Correct ligations for vector construction are confirmedaccording to the procedures of Young et al., supra.

cDNA Library Screening

cDNA libraries can be screened using reduced stringency conditions asdescribed by Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Greene Publishing and Wiley-Interscience, New York (1990), or by usingmethods described in Sambrook et al. supra), or by using a colony orplaque hybridization procedure with a fragment of the DOR-1 cDNA codingfor opioid receptor protein.

Plaque hybridization is typically carried out as follows: Host bacteriasuch as LE 392 (Stratagene) are grown overnight at 37° in LB Broth(Sambrook et al., supra), gently pelleted and resuspended in one halfthe original volume of 10 MM MgSO₄, 10 mM CaCl₂. After titration, anamount of the phage library containing approximately 50,000 plaqueforming units (pfu) is added to 300 μl of the host bacteria, incubatedat 37° for 15 minutes and plated onto NZYCM agar with 10 ml NZYCM topagarose. A total of a million plaques distributed on twenty 15 cm platesare screened. For colony screening, transfected bacteria are plated ontoLB broth plates with the appropriate antibiotics. After the plaques orcolonies have grown to 1 mm, the plates are chilled at 4° C. for atleast two hours, and then overlaid with duplicate nitrocellulosefilters, followed by denaturation of the filters in 0.5 M NaOH/1.5 MNaCl for five minutes and neutralization in 0.5 M Tris, pH 7.4/1.5 MNaCl for five minutes. The filters are then dried in air, baked at 80°C. for two hours, washed in 5×SSC/0.5% SDS at 68° C. for several hours,and prehybridized in 0.5 M NaPO₄, pH 7.2/1% BSA/1 mM EDTA/7% SDS/100μg/ml denatured salmon sperm DNA for more than 4 hours. Using the DOR-1cDNA (described herein) labeled by random priming as a probe, highstringency hybridization is carried out in the same solution at 68° C.,and the temperature is reduced to 50-60° C. for lower stringencyhybridization. After hybridization for 16-24 hours, the filters arewashed first in 40 mM NaPO₄, pH 7.2/0.5% BSA/5% SDS/1 mM EDTA twice forone hour each, then in 40 mM NaPO₄, pH 7.2/1% BSA/1 mM EDTA for one houreach, both at the same temperature as the hybridization (Boulton et al.,Cell (1991) 65:663-675). The filters are then exposed to film with anenhancing screen at −70° C. for one day to one week.

Positive signals are then aligned to the plates, and the correspondingpositive phage is purified in subsequent rounds of screening, using thesame conditions as in the primary screen. Purified phage clones are thenused to prepare phage DNA for subcloning into a plasmid vector forsequence analysis. Tissue distribution of DNA corresponding to thevarious independent clones is analyzed using Northern blots and in situhybridization using standard methods. Function of the DNA is testedusing expression in a heterologous eucaryotic expression system such asCOS cells.

Expression of Opioid Receptor Protein

The nucleotide sequence encoding opioid receptor protein can beexpressed in a variety of systems. The cDNA can be excised by suitablerestriction enzymes and ligated into procaryotic or eucaryoticexpression vectors for such expression.

For example, as set forth below, the cDNA encoding the protein isexpressed in COS cells. To effect functional expression, the plasmidexpression vector CDM8 (Aruffo and Seed, Proc Natl Acad Sci USA (1987)84:8573-8577, provided by Drs. Aruffo and Seed (Harvard University,Boston, Mass.) was used. Alternatively, other suitable expressionvectors such as retroviral vectors can be used.

Procaryotic and preferably eucaryotic systems can be used to express theopioid receptor. Eucaryotic microbes, such as yeast, can be used ashosts for mass production of the opioid receptor protein. Laboratorystrains of Saccharomyces cerevisiae, Baker's yeast, are used most,although a number of other strains are commonly available. Vectorsemploying, for example, the 2μ origin of replication (Broach, Meth Enz(1983) 101:307), or other yeast compatible origins of replications(e.g., Stinchcomb et al., Nature (1979) 282:39); Tschempe et al., Gene(1980) 10:157; and Clarke et al., Meth Enz (1983) 101:300) can be used.Control sequences for yeast vectors include promoters for the synthesisof glycolytic enzymes (Hess et al., J Adv Enzyme Reg (1968) 7:149;Holland et al., Biochemistry (1978) 17:4900). Additional promoters knownin the art include the promoter for 3-phosphoglycerate kinase (Hitzemanet al., J Biol Chem (1980) 255:2073), and those for other glycolyticenzymes. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and enzymesresponsible for maltose and galactose utilization. It is also believedterminator sequences are desirable at the 3′ end of the codingsequences. Such terminators are found in the 3′ untranslated regionfollowing the coding sequences in yeast-derived genes.

Alternatively, genes encoding opioid receptor protein are expressed ineucaryotic host cell cultures derived from multicellular organisms.(See, e.g., Tissue Cultures, Academic Press, Cruz and Patterson, eds,(1973)). These systems have the additional advantage of the ability tosplice out introns, and thus can be used directly to express genomicfragments. Useful host cell lines include amphibian oocytes such asXenopus oocytes, COS cells, VERO and HeLa cells, Chinese hamster ovary(CHO) cells, and insect cells such as SF9 cells. Expression vectors forsuch cells ordinarily include promoters and control sequences compatiblewith mammalian cells such as, for example, the commonly used early andlate promoters from baculovirus, vaccinia virus, Simian Virus 40 (SV40)(Fiers et al., Nature (1973) 273:113), or other viral promoters such asthose derived from polyoma, Adenovirus 2, bovine papilloma virus, oravian sarcoma viruses. The controllable promoter, hMTII (Karin et al.,Nature (1982) 299:797-802) may also be used. General aspects ofmammalian cell host system transformations have been described by Axel,U.S. Pat. No. 4,399,216. It now appears, that “enhancer” regions areimportant in optimizing expression; these are, generally, sequencesfound upstream or downstream of the promoter region in non-coding DNAregions. Origins of replication can be obtained, if needed, from viralsources. However, integration into the chromosome is a common mechanismfor DNA replication in eucaryotes.

If procaryotic systems are used, an intronless coding sequence should beused, along with suitable control sequences. The cDNA of opioid receptorprotein can be excised using suitable restriction enzymes and ligatedinto procaryotic vectors along with suitable control sequences for suchexpression.

Procaryotes most frequently are represented by various strains of E.coli; however, other microbial species and strains may also be used.Commonly used procaryotic control sequences which are defined herein toinclude promoters for transcription initiation, optionally with anoperator, along with ribosome binding site sequences, including suchcommonly used promoters as the β-lactamase (penicillinase) and lactose(lac) promoter systems (Chang et al., Nature (1977) 198:1056) and thetryptophan (trp) promoter system (Goeddel et al., Nucl Acids Res (1980)8:4057) and the λ derived P_(L) promoter and N-gene ribosome bindingsite (Shimatake et al., Nature (1981) 292:128).

Depending on the host cell used, transformation is carried out usingstandard techniques appropriate to such cells. The treatment employingcalcium chloride, as described by Cohen, Proc Natl Acad Sci USA (1972)69:2110 (1972) or by Sambrook et al. (supra), can be used forprocaryotes or other cells which contain substantial cell wall barriers.For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology (1978) 54:546,optionally as modified by Wigler et al., Cell (1979) 16:777-785, or byChen and Okayama, supra, can be used. Transformations into yeast can becarried out according to the method of Van Solingen et al., J Bact(1977) 130:946, or of Hsiao et al., Proc Natl Acad Sci USA (1979)76:3829.

Other representative transfection methods include viral transfection,DEAE-dextran mediated transfection techniques, lysozyme fusion orerythrocyte fusion, scraping, direct uptake, osmotic or sucrose shock,direct microinjection, indirect microinjection such as viaerythrocyte-mediated techniques, and/or by subjecting host cells toelectric currents. The above list of transfection techniques is notconsidered to be exhaustive, as other procedures for introducing geneticinformation into cells will no doubt be developed.

Modulation of Expression by Antisense Sequences

Alternatively, antisense sequences may be inserted into cells expressingopioid receptors as a means to modulate functional expression of thereceptors encoded by sense oligonucleotides. The antisense sequences areprepared from known sense sequences (either DNA or RNA), by standardmethods known in the art. Antisense sequences specific for the opioidreceptor gene or RNA transcript can be used to bind to or inactivate theoligonucleotides encoding the opioid receptor.

Terminology

As used herein, the singular forms “a”, “an” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to “a receptor” includes mixtures of such receptors,reference to “an opioid” includes a plurality of and/or mixtures of suchopioids and reference to “the host cell” includes a plurality of suchcells of the same or similar type and so forth.

Unless defined otherwise all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The following examples areintended to illustrate but not to limit the invention. Temperatures arein ° C. and pressures at near atmospheric unless otherwise specified.

Preparation of Mono ¹²⁵I-DADLE

DADLE (Peninsula Laboratories Inc.) was iodinated using the iodogenmethod (Maidment et al., in: MICRODIALYSIS IN THE NEUROSCIENCES, T.Robinson and J. Justice, eds., pp. 275-303 (Elsevier, 1991)). Both mono-and di-iodinated forms are produced. It has been reported thatdi-iodo-DADLE does not bind opiate receptors, due to the di-iodinationof the tyrosine residue (Miller, R. J., et al., Life Sci (1978)22:379-88). Accordingly, mono-iodinated DADLE is preferred.Mono-¹²⁵I-DADLE is also preferred because it has extremely high specificactivity compared to DADLE labeled with other isotopes. Thus, exposuretimes on the order of days, rather than weeks or months can be used.

By employing a molar ratio of sodium iodide to peptide of approximately1:100 when carrying out iodination, the yield of the preferredmono-iodinated DADLE was increased. Additionally, to further enhance theyield of the mono-iodinated form, iodinated DADLE (containing both mono-and di-iodinated forms) was purified by reverse-phase HPLC (Maidment etal., supra). Employing this procedure a single major radiolabeled peakof the mono-iodinated DADLE separated from di-iodinated andnon-iodinated forms.

DADLE monolabeled with ¹²⁵I is crucial to successful screening.Radiolabeled ¹²⁵I-DADLE differs from DADLE in several importantparameters: size, hydrophobicity, and binding affinity (slightly lower).The purification of mono-iodinated from di-iodinated and non-iodinatedDADLE by the HPLC step yields a ligand with very high specific activity(approximately 2000 Ci/mmol). The specific activity of themono-iodinated form is approximately 100 times greater than thatobtained by using the unseparated mixture of mono-, di-, andnon-iodinated DADLE. Monolabeled 125I-DADLE must be used within a fewdays of its preparation.

EXAMPLE 1 Preparation of DOR-1

The NG108-15 cell line (available from Dr. Christopher Evans, UCLA)comprises a homogeneous and enriched source of delta opioid receptors.Utilizing mRNA isolated from NG108-15, a random-primed, size-selectedcDNA library was constructed in plasmid vector CDM8. The cDNA librarywas amplified in bacteria. The cDNA library was transfected into COS-7cells by electroporation. Transiently transfected COS lawns werescreened and selected with highly purified mono-¹²⁵I-2dAla, 5dLeuenkephalin (¹²⁵I-DADLE). Positive clones were identified by filmautoradiography, and plasmids from these cells were recovered andamplified in bacteria. Thereafter, the plasmids were re-transfected intoCOS cells. Following three cycles of such plasmid enrichment, individualclones were transfected and a pure clone was identified that bound¹²⁵I-DADLE.

A. Construction of the cDNA Library

RNA was prepared from NG108-15 cells by homogenization in 6 Mguanidinium isothiocyanate, followed by centrifugation through cesiumchloride (J. M. Chirgwin, et al., Biochemistry (1979) 18:5294). Poly-A⁺RNA was isolated by chromatography over oligo-dT-cellulose (H. Aviv andP. Leder, Proc Natl Acad Sci USA (1972) 69:1408). Using this RNA as atemplate, random hexamers were used to prime cDNA synthesis by avianmyeloblastosis virus reverse transcriptase (Life Sciences Inc.). Secondstrand synthesis was accomplished with RNase-H and E. coli DNApolymerase (U. Gubler and B. J. Hoffman, Gene (1983) 24:263). The endsof the cDNAs were rendered blunt with T4 DNA polymerase and BstXIlinkers were added. cDNA longer than 1.5 kb was selected byelectrophoresis through 5% acrylamide followed by electro-elution. The1.5 kb cDNA was ligated to the CDM8 vector (A. Aruffo and B. Seed,supra, and then transformed into MC-1061 bacteria by electroporation (W.J. Dower et al., Nucl Acids Res (1988) 16:6127). Accordingly, six poolsof plasmid DNA were prepared from the original cDNA library ofapproximately 2×10⁶ recombinants.

B. Plasmid Transfection by Electroporation and Expression in COS Cells

COS cells were grown at high density and were harvested in trypsin, thenresuspended at 2×10⁷/ml in 1.2×RPMI containing 20% fetal calf serum.These cells were then incubated for ten minutes at 4° C. with 20 μgrecombinant plasmid DNA from the cDNA library described above, and thenelectroporated at 960 μF and 230 V in a 0.4 cm gap cuvette (BioRad). Thecells were then incubated an additional ten minutes at 4° C., and thenplated into Dulbecco's Modified Eagle's Medium (DMEM) plus 10% fetalcalf serum (FCS).

C. Screening of Transfected COS Cells

The transfected COS cells as obtained above were grown for three days,then screened using radiolabeled mono ¹²⁵I-DADLE. Transfected COS lawnswere washed with PBS, then incubated at room temperature with 10-20 nM¹²⁵I-DADLE in KHRB containing 1% BSA. After 1 hour, the plates werewashed rapidly several times with ice cold PBS then dried on ice withstrong flow of forced cold air. Plates were exposed on Dupont Cronexfilm in cassettes at room temperature. Positive clones were identifiedby careful alignment of the film with the petri dish via low powermicroscopy.

DNA was removed from positive cells by solubilization in 0.1% SDS in TEcontaining 1 μg/μl tRNA delivered from a syringe attached to a capillarytube on a micromanipulator. Plasmids were purified from the extractedcells using the Hirt lysis procedure (Hirt, B., J Mol Biol (1967)26:365-369), and electroporated into MC-1061 bacteria. The plasmids werepurified then retransfected into COS cells. Following three suchenriching cycles, individual plasmid clones were transfected into COScells yielding a single clone, named the DOR-1 clone.

EXAMPLE 2 Characterization of DOR-1

The DOR-1 clone initially was characterized by screening cell membranefractions, from cells expressing DOR-1, with the labelled DADLE it wasfound that binding of ¹²⁵I-DADLE was displaced by nanomolarconcentrations of opiate alkaloids diprenorphine, morphine, etorphine,and by DADLE, DSLET and DPDPE. Dextrorphan (10 μM) did not displace the¹²⁵I-DADLE, whereas its opioid-active enantiomer levorphanol diddisplace the radiolabeled DADLE. Additionally, the mu receptor-selectiveligand DAGO (5 μM) did not displace the counts.

The DOR-1 clone was further characterized pharmacologically by assessingbinding of ³H-diprenorphine to intact cells expressing the DOR-1 clone(FIG. 1), and by assessing displacement of ³H-diprenorphine frommembrane fractions of such cells (FIGS. 2 and 3).

Binding assays were conducted on intact cells in KRHB, 1% BSA; or onmembranes in 25 mM HEPES, 5 mM MgCl₂ pH 7.7. Cells were harvested withPBS containing 1 mM EDTA, washed 2× with PBS then resuspended in KHRB.Membranes prepared from the cells (Law P. Y. E et al., Mol Pharm (1983)23:26-35) were used directly in the binding assay. Binding assays wereconducted in 96 well polypropylene cluster plates (Costar), at 4° C. ina total volume of 100 μl with an appropriate amount of radiolabeledligand. Following 1 hour of incubation, plates were harvested on aTomtec harvester and “B” type filtermats were counted in a Betaplate(Pharmacia) scintillation counter using Meltilex B/HS (Pharmacia)melt-on scintillator sheets.

Intact cells expressing DOR-1 were analyzed with the high affinityopiate antagonist ³H-diprenorphine. Specific binding was defined by thecounts displaced by 400 nM diprenorphine. FIG. 1 shows a saturationcurve for ³H-diprenorphine for NG108-15 cells, and COS-7 cellstransfected with the delta opioid receptor clone. Untransfected COScells, or COS cells transfected with plasmid having no insert showed nospecific binding. Thus, the opioid binding of COS-DOR-1 cells wassimilar to that of NG108-15 cells.

Membranes prepared by standard methods from transfected COS-7 cells wereemployed for a more extensive pharmacological characterization of thereceptor encoded by the DOR-1 clone. The affinities for the followingalkaloid opiates in competition for ³H-diprenorphine are illustrated inFIG. 2: unlabeled diprenorphine, a high affinity antagonist for deltareceptors; etorphine, a high affinity agonist for delta, mu and kappareceptors; levorphanol, a low affinity agonist for delta receptors;morphine, a low affinity agonist for delta receptors and a high affinityagonist for mu receptors; and dextrorphan, a non-opiate activeenantiomer of levorphanol which should not bind delta receptors.

As shown in FIG. 2, the displacement of ³H-diprenorphine, in decreasingorder of affinity, was observed with diprenorphine, etorphine,levorphanol and morphine. As expected, ³H-diprenorphine was notdisplaced by dextrorphan.

The affinities of the following opioid peptides in competition for³H-diprenorphine are set forth in FIG. 3: DADLE, a high affinity agonistfor mu and delta receptors; DSLET and DPDPE, both high affinity agonistsof delta (but not mu) receptors; DAGO, a selective agonist for mureceptors; and Dynorphin 1-17, a high affinity agonist for kappareceptors and moderate to low affinity agonist for delta receptors. Asshown in FIG. 3, the displacement of ³H-diprenorphine, in decreasingorder of affinity, was observed for DSLET, DPDPE and DADLE, andDynorphin 1-17. Only weak displacement by DAGO was observed.

EXAMPLE 3 Northern Blot Analysis of RNA

For Northern analysis, the mRNA from NG108-15 cells, and from cellsdissected from regions of rat brain was separated by electrophoresisthrough 2.2 M formaldehyde/1.5% agarose, blotted to nylon and hybridizedin aqueous solution at high stringency. The filters were prehybridizedin 0.5 M NaPO₄, pH 7.2; 1% BSA; 1 mM EDTA; 7% SDS; and 100 μg/mldenatured salmon sperm DNA for at least four hours at 68° C. (Boulton etal., supra). The filters were then hybridized overnight under these sameconditions with ≧5×10⁶ cpm/ml purified cDNA insert labelled by randompriming (A. P. Feinberg and B. Vogelstein, Anal Biochem (1983) 132:6).The filters were twice washed in 40 mM NaPO₄, pH 7.2; 0.5% BSA; 5% SDS;and 1 mM EDTA for one hour, and then washed twice in 40 mM NaPO₄, pH7.2; 1% SDS; and 1 mM EDTA for one hour each, all at 68° C. Thereafterautoradiography was performed with DuPont Cromex Lightening Plus at −70°C.

The results of the Northern analysis of the mRNA showed the presence ofmultiple bands hybridizing to the probe at approximately 8.7, 6.8, 4.4,2.75 and 2.2 kilobases (Kb) (FIG. 4). Also, the Northern analysisindicates that the pattern of mRNA may vary between brain regions. Atpresent, it is unclear whether these mRNAs encode different proteinsequences, and if so, whether these messages represent different typesor sub-types of opioid receptors.

EXAMPLE 4 Southern Blot Analysis of DNA

The radiolabeled DOR-1 cDNA probe was hybridized to genomic Southernblots by standard methods (Sambrook et al., supra). Accordingly, theradiolabeled DOR-1 cDNA probe was hybridized under high stringencyconditions to a blot of NG108-15, mouse, rat and human DNA cut withrestriction endonuclease BamHI (FIG. 7). Single bands were observed inthe clones containing the NG108-15, mouse, and rat DNA. The sizes of thebands hybridizing to the cDNA probe were estimated to be 5.2 kb(NG108-15), 5.2 kb (mouse), and 5.7 kb (rat). These results indicate theclose homology of the mouse and rat genes, and also demonstrate that theDOR-1 clone is from the murine parent of the NG108-15 cell line.

In a blot containing EcoRI-cut genomic DNA from many different species,hybridization of the DOR-1 cDNA under conditions of moderate stringencyshowed two bands in each lane of mouse, rat, human, rabbit, and severalother mammalian species. This demonstrates a close relationship betweenopioid receptor genes in all of these species. Further, these resultsshow that the genes or cDNAs from each of these species may readily becloned using hybridization under moderate stringency.

EXAMPLE 5 Determination of the cDNA Sequence

Isolated cDNA represented by the DOR clone was analyzed by subcloningthe insert from the cDNA clone into a plasmid such as pBluescript™(Stratagene, San Diego, Calif.) and using the dideoxy method (Sanger etal., Proc Natl Acad Sci USA (1977) 74:5463-5467). The sequence of thecDNA was determined from single-stranded DNA and specifically designedinternal primers, using both Sequenase and ΔTaq cycle sequencing kits(USB). These kits, widely used in the art, utilize the dideoxy chaintermination method. The DNA sequence and predicted protein sequence wasthen compared to sequences in established databanks such as GenBank.

Sequencing the cDNA insert in the DOR-1 clone, revealed an open readingframe of 370 amino acids (FIG. 5). Comparisons with known sequences inGenBank showed highest homology between DOR-1 and the G-protein-coupledsomatostatin receptor (57% amino acid identity), and slightly lowerhomology with the receptors binding angiotensin, the two chemotacticfactors IL-8 and N-formyl peptide. FIG. 6 shows the homology to thehuman somatostatin 1 receptor. The close homology of the presentreceptor clone with the somatostatin receptor is especially noteworthysince somatostatin ligands are reported to bind to opioid receptors, andto have molecular mechanisms similar to those in delta receptors.

Other features of the DOR-1 clone amino acid sequence deduced from thecDNA sequence include three consensus glycosylation sites at residues 18and 33 (predicted to be in the extracellular N-terminal domain) and atresidue 310 (close to the C-terminus and predicted to be intracellular).Phosphokinase C consensus sites are present within predictedintracellular domains, at residues 242, 255, 344, and 352. Sevenputative membrane-spanning regions were identified based onhydrophobicity profiles, as well as homology with Rhodopsin and otherG-protein coupled receptors which have been analyzed with respect tomembrane-spanning regions using Macvector (I.B.I.) analysis. The DOR-1clone isolated in accordance with the principles of the presentinvention produces a delta receptor with a predicted molecular weight of40,558 daltons prior to post-translational modifications such asN-glycosylation.

EXAMPLE 6 Isolation of Opioid Receptor Genomic Clones

Isolation of genomic clones was carried out according to techniquesknown in the art. To isolate opiate receptor genomic clones, 300,000human genomic clones in γ-gem 11 (Promega) and a similar number of mousegenomic clones in lambda Fix (Stratagene) were plated on host strainLe392 and probed with the 1.1 kb DOR-1 Pst/Xba I fragment, whichcontains primarily the coding region. The conditions for hybridizationwere of fairly low stringency: 50% formamide/6×SSC, overnight at 37° C.The washes were performed also at low stringency: 2×SSC, 0.1% SDS atroom temperature.

One mouse clone and three human genomic clones were isolated andpurified by sequential rounds of hybridization and plaque purification.DNA preparation and restriction analysis showed that the three humanclones had very different EcoRI digestion patterns. The 1.1 kb opiatereceptor probe hybridized to a different single EcoRI band in Southernblot analysis for each clone. These results indicated preliminarily thatthree different genes were represented by the human genomic clones whichwere designated H3, H14 and H20 (see FIGS. 8 a, 8 b, 8 c and 8 d). Eachof these clones was deposited on Aug. 13, 1993 at the American TypeCulture Collection, Rockville, Md., under conditions of the BudapestTreaty. All restrictions on access to these deposits will be irrevocablyremoved at the time a patent issues in the United States on the basis ofthis application. The ATCC deposit numbers are 75551 for H3 (.delta.),75550 for H14 (.kappa.), and 75549 for H20. (.mu.).

The H3, H14 and H20 clones were digested into smaller fragments by EcoRIand TaqI and then shotgun cloned into the appropriate site of Bluescriptfor sequencing. The partial nucleotide sequence for H3 is shown in FIG.8 a; the partial nucleotide sequence of H14 is shown in FIG. 8 b; thepartial nucleotide sequence of H20 is shown in FIG. 8 c.

The three genomic clones were mapped by in situ hybridization on humanmetaphase chromosomes by Dr. Glenn Evans of the Salk Institute. H3 mapsto chromosome 1P; H14 maps near the centromere of chromosome 8, and H20maps to chromosome 6. Comparison of sequence data obtained as describedabove with the published sequences for the murine counterpartsreferenced hereinabove, and with the DOR-2 clone described hereinbelow,confirmed that: (a) H3 encodes the human delta opioid receptor; (b) H14encodes the human kappa opioid receptor and (c) H20 encodes the human mureceptor. In addition, H20 appears to contain a CACACA marker (FIG. 8 d)which provides a means to track the inheritance of this gene.

The genomic clones were digested into smaller fragments by EcoRI andTaqI, then shotgun cloned into the appropriate site of Bluescript forsequencing.

EXAMPLE 7 Isolation of Opioid Receptor Clones from Additional Organisms

In order to isolate the opioid receptor from mammalian brain cells, forexample human brain cells, a random-primed human brainstem cDNA libraryin λ Zap (Stratagene) was screened using the murine cDNA encoding theDOR-1 described herein. Positive plaques were purified and rescreened.Individual positive clones are sequenced and characterized as above.

EXAMPLE 8 Determination of Probable Antigenic Sequences

By evaluating the amino acid sequence of the opioid receptor encoded byDOR-1 with the MacVector (I.B.I.) antigenic index, and the antigenicindex in accordance to Jameson, B. and H. Wolf, Comput Applic in Biosci(1988) 4:181-186, the following underlined sequences of the delta opioidreceptor were determined to have a high antigenic potential:

NH₂ MELVPSARAELQSSPLVNLSDAFPSAFPSAGANASGSPGARSASSLALAIAITALYSAVCAVGLLGNVLVMFGIVRYTKLKTATNIYIFNLALADALATSTLPFQSAKYLMETWPFGELLCKAVLSIDYYNMFTSIFTLTMMSVDRYIAVCHPVKALDFRTPAKAKLINICIWVLASGVGVPIMVMAVTQPRDGAVVCMLQFPSPSWYWDTVTKICVFLFAFVVPILIITVCYGLMLLRLRSVRLLSGSKEKDRSLRRITRMVLVVVGAFVVCWAPIHIFVIVWTLVDINRRDPLVVAALHLCIALGYANSSLNPVLYAFLDENFKRCFRQLCRTPCGRQEPGSLRRPRQATTRERVTACTPSDGPGGGAAA-COOH (SEQ ID NO: 2).The N-terminal sequence is extracellular, the other four sequences arepredicted to be intracellular.

EXAMPLE 9 Recovery of the Murine Clone DOR-2 (mMOR-1)

A cDNA library prepared from mouse brain in λgt10 was probed using thelow-stringency conditions of Example 6 using DOR-1 as a probe. One clonewas recovered, inserted into Bluescript and sequenced. Northern andSouthern blots indicated divergence from DOR-1. This clone, designatedDOR-2, represented a new gene. DOR-2 hybridized to a different patternof neurons than did DOR-1 and showed greater labeling of the striatum.Expression of DOR-1 by insertion into the vector pCDNA and transfectioninto mammalian cells produced cells which bind morphine, indicative of amu-receptor. The cells also bind the nonselective opiate antagonistdiprenorphine. The identity of DOR-2 (mMOR-1) as that of a mu receptorwas confirmed by the displacement of ³H-DPN by nanomolar concentrationsof the mu-selective ligands morphiceptin, DAMGO and morphine. The deltaselective ligands DPDPE and deltorphan did not displace the binding andnaloxone had the expected high affinity. The partial sequence designatedH20, described in Example 6, was substantially similar to DOR-2. Thepartial sequence of DOR-2 is shown in FIG. 9.

FIG. 10 shows a comparison of the amino acid sequences of murine deltareceptin with the rat mu and kappa receptors. There are extensiveregions of homology.

EXAMPLE 10 Isolation of ORL-1

A human brain stem cDNA library was obtained from Stratagene and probedusing low-stringency hybridization with the murine DOR-1 sequence shownin FIG. 5 under stringency conditions of 50% formamide/6×SSC at 37° C.with washes of 1×SSC/0.1% SDS at 37° C. A partial cDNA clone encodingORL-1 was obtained and completed at the 5′ end by RACE using cDNAobtained from human brain. The DNA sequence obtained for ORL-1 is shownin FIG. 11 and is identical to that reported by Mollereau et al.(supra). ORL-1 has approximately 44% amino acid identity to the mureceptor.

In addition to ORL-1, three clones for ORL-2 were obtained and afull-length clone was assembled from two overlapping clones. Thesequence of one of the ORL-2 clones was identical to that reported byO'Dowd et al. (supra) while the other had a base change at Leu¹²⁹ whichdid not result in an alteration of amino acid sequence.

FIG. 12 compares the protein sequences of three cloned opioid receptorsand ORL-1 and ORL-2.

Multiple PKC and PKA cites in the third intracellular loop of ORL-1 aresimilar to those in the delta opioid receptor. However, a His residuepresent in the sixth transmembrane domain of all the opioid receptors isabsent in ORL-1; this His residue may play a role in aromaticinteraction with ligands and may be critical for opioid receptorbinding.

Mollereau et al. (supra) have shown that a stable cell line transfectedwith ORL-1 shows etorphinne-induced cyclase inhibition. This inhibitionis reversible with diprenorphine, although labeled diprenorphine bindingto ORL-1 has not been shown. In addition, ORL-1 has two Asn-linkedglycosylation sites in the N-terminal extracellular domain as shown inFIG. 12.

1. An isolated protein which is functional as a delta opioid receptor,wherein said protein is encoded by a nucleotide sequence that hybridizesunder conditions of stringency corresponding to washing at 5×SSC/0.5%SDS at 68° C. to the full-length complement of a nucleotide sequenceencoding DOR-1 (SEQ ID NO: 8).
 2. The protein of claim 1, which has theamino acid sequence of DOR-1 (SEQ ID NO: 8).
 3. The protein of claim 1,which is prepared by expressing the nucleotide sequence encoding saidprotein in a heterologous recombinant host.
 4. A method to identify acompound that binds to delta opioid receptor wherein said methodcomprises contacting the protein of claim 1 with a candidate compoundand assessing the binding of said candidate compound to said protein,whereby a compound that binds said protein is identified as a compoundthat binds delta opioid receptor.
 5. The method of claim 4, wherein saidassessing comprises measuring the ability of said candidate compound tocompete with a labeled delta opioid ligand for binding to said protein.